Our long-term objective is to understand the functions of accessory proteins of primate lentiviruses at the molecular level. Accessory proteins are important virulence factors that modify cellular environments to be more conducive for replication of these viruses in the host. Vpr is a small multifunctional adaptor protein, that is encoded by all primate lentiviruses and interferes with DNA metabolism and cell cycle progression in the infected cells. This leads to their arrest in late S/early G2 phase at the DNA damage checkpoint and activates apoptotic program. Vpx, a factor closely related to Vpr, is required for the ability of HIV-2 and SIVsm/mac viruses to replicate in macrophages. Although Vpr and Vpx are important virulence factors, the molecular mechanisms that mediate their functions are not well understood. To gain new insights into mechanisms exploited by Vpr and Vpx, they were immuno-affinity purified from cells and their associated proteins identified by multidimensional chromatography-coupled tandem mass spectroscopy (MudPIT). These studies revealed that Vpr and Vpx specifically and abundantly associate with a protein complex comprising subunits of a E3 ubiquitin ligase assembled on Cullin-4 scaffold (Cul4-DDB1[VprBP]), which we linked to the control of cell cycle and DNA replication, and identified additional cellular proteins targeted by Vpr and Vpx. Importantly, Vpr and Vpx appear to regulate ubiquitin ligase activity of the Cul4 E3 complex they bind. This property correlates with the ability of Vpr to arrest cells in G2. Together evidence suggests that Vpr and Vpx carry out their functions by usurping the Cul4-DDB1[VprBP] E3 ubiquitin ligase to modulate ubiquitination of specific but distinct sets of cellular proteins. Therefore understanding the interactions of these virulence factors with the Cul4-DDB1[VprBP] E3 ligase complex and identification of cellular proteins whose ubiquitination they alter is required for the understanding of their functions at the molecular level. Towards these goals the first specific aim will characterize the regulation of Cul4- DDB1[VprBP] E3 complex by Vpr and Vpx accessory factors. The second specific aim will assess the role of Vpx-associated VprBP, and its associated Cul4 E3, for its ability to facilitate macrophage infection. The roles of other select Vpx-associated proteins identified using mass spectroscopy will also be addressed. The third specific aim will identify cellular proteins recruited by Vpr and VprBP for ubiquitination by Cul4, by using a combination of biochemical purification techniques and MudPIT. These studies will provide a framework for understanding the molecular interactions that underlie the functions of Vpr and Vpx, how they usurp Cul4-DDB1[VprBP] E3 ubiquitin ligase to facilitate the replication cycle of primate lentiviruses and will have implications for rational design of effective strategies to disrupt their functions. PUBLIC HEALTH RELEVANCE: Accessory proteins of primate lentiviruses, such as Vpr and Vpx, are important virulence factors. The proposed studies are aimed at understanding molecular mechanism that these proteins use to facilitate replication of primate lentiviruses and may provide the basis for developing therapeutic agents that will delay development of AIDS.